David Kristofferson

Microtubule assembly: a review of progress, principles, and perspectives

Publisher Summary Substantial improvements in electron, light, and fluorescence microscopy, as well as the isolation of discrete protein components of the cytoskeleton, have led the way to a much better appreciation of the structural organization of the cytoplasm. Indeed, the lacelike network of thin filaments, intermediate filaments, and microtubules in nonmuscle cells is as familiar today as the organelles identified after the advent of biological electron microscopy. A major means for characterizing and comprehending the emerging principles of cytoskeletal action has been to isolate and define the properties of various constituents. This chapter considers one such component, the microtubule, from the perspective of the theoretical and experimental efforts to elucidate its assembly and disassembly properties. For this purpose, it concentrates on the in vitro self-assembly of microtubules derived from the neural systems.

Time scale of microtubule length redistribution

Abstract This report presents electron microscopic evidence of statistically significant changes in the microtubule number concentration and length distribution after the attainment of monomer-polymer equilibrium (“steady state”). We also extend previous theoretical work on polymer redistribution ( F. Oosawa, 1970, J. Theor. Biol. 27 , 69–86 ).

Microtubule interactions with GDP provide evidence that assembly-disassembly properties depend on the method of brain microtubule protein isolation☆

Abstract Microtubule stability in the presence of GDP was found to depend on the microtubule protein purification method used, even though all are variations on the use of warm-induced assembly and cold-induced disassembly with intervening centrifugation steps. Substantial differences in the assembly-disassembly properties were observed with protein prepared in the absence or presence of glycerol under hypotonic extraction conditions as compared to the microtubule protein from a sucrose extraction protocol which minimizes contamination resulting from osmotic shock of cellular organelles. Assembled protein from either hypotonic preparation partially maintained its state of assembly after GDP addition. With protein from the sucrose method, assembled tubules are almost completely depolymerized upon enzymatic conversion of GTP to GDP. The characteristics of the elongation reaction following addition of GDP to assembling microtubule protein also depend on the isolation method. We discuss the conflicting claims in the literature on microtubule interactions with GDP in the light of these new findings.

A quantitative treatment of reversible monomer-polymer exchange reactions with microtubules and other biopolymers☆

Abstract We present a quantitative solution to the problem of equilibrium exchange of radioactive monomers into biological polymers. This solution avoids approximations used in earlier attempts to handle the problem. The analysis therefore provides a better theoretical basis for evaluating data for head-to-tail polymerization in biopolymers such as F-actin and microtubules. A discussion of the models implications concerning head-to-tail polymerization and an extension of the analysis to drug action on biopolymer assembly is included.

Chapter 8 An Automated Method for Defining Microtubule Length Distributions

Publisher Summary This chapter presents two examples of automated data acquisition and evaluation and examines various statistical problems, with examples of actual experiment data and a detailed program listing for implementing this automated method. Length distribution measurements often provide the only available way to determine important parameters in the study of biological polymers. For example, because most polymerization mechanisms tend to be endwise, it is necessary to know the number of polymer ends in solution to study polymerization or depolymerization kinetics. This can be determined from knowledge of the total protein in polymer form (usually determined by turbidity) and the average polymer length. The initial rates determined by turbidity have agreed with the electron microscopic number concentration measurements to within a few percent. Accurate determinations of microtubule distribution shapes have enabled theoretically predicting the entire time course of the depolymerization reaction on the basis of these electron microscopic measurements. These results demonstrate that electron microscopy can be used to accurately determine microtubule lengths in solution. In addition, length distribution techniques may be used to determine the mode of action of other microtubule assembly effectors.

[42] Microtubule disassembly: A quantitative kinetic approach for defining endwise linear depolymerization

Publisher Summary This chapter presents the pertinent theoretical aspects and describes the experimental approaches to characterize the mechanism of depolymerization. To analyze endwise depolymerization quantitatively, various premises must hold: (1) the polymer undergoes stepwise disassembly in a series first-order fashion, (2) the off-rate constant is independent of polymer length over the course of depolymerization, (3) the on-rate constant is zero, (4) turbidity is a measure of the remaining polymer weight concentration and is independent of the polymer length distribution, and (5) the concentrations of the various polymer lengths may be estimated by use of electron microscopy. Primarily, the chapter examines the kinetics of the series first-order decay of polymer and its expression in terms of remaining polymer weight concentration. For indefinite polymerization processes that result from entropydriven condensation equilibria, there are three ways to effect depolymerization: (1) dilution to below the critical concentration, (2) reduction of the temperature to destabilize the polymer, and (3) addition of a reagent to reduce the concentration of the form of the protomer in equilibrium with the polymer. The methods for bringing about such changes must be rapid relative to the time course of depolymerization; otherwise, the kinetics of effecting the depolymerization process will obscure the kinetics of polymer loss.